Hyperphosphorylatedtauaggregation-basedAlzheimer?sdiseaseearlydrugdiscovery Alzheimer?s disease (AD) is an irreversible neurodegenerative disease affecting 47 million people worldwide, and costs 604 billion US dollars every year for medical expenses. To date, there is no cure or prevention for AD. The two defining features of AD are Ab? plaques and neurofibrillary tangles that are composed of hyperphosphorylatedtau.WhiletheADdrugdiscoverylandscapehasbeendominatedbyanti-Ab?measures, recurringfailuresofclinicaltrialsarguestronglythatarealignmentofthedrugtargetandstrategiesisneededto makeabreakthroughinADtherapeuticsdevelopment.Indeed,multiplelinesofevidencesuggestthatthepre- tangle stage of hyperphosphorylated tau aggregates cause diffusible cytotoxicity that likely underlies neurodegeneration.Screeningforcompoundsthatpreventhyperphosphorylatedtaufromformingthecytotoxic aggregatesthusaffordsamoreviablerouteforADdrugdiscovery. Thetangle-centricdrugdesignhasnotcometofruition.Awidespectrumofcompoundshavebeen identified in multiple screens as inhibitors of tangle formation,but later found to be false-positive.One shared issueforthesescreensistheuseofanunmodifiedtauproteinthatrequiresaninducer,e.g.,heparin,forefficient aggregation in a reducing environment. The assay subject (tau) lacks the pathological mark of hyperphosphorylation,andthediseaserelevanceoftheinducerhasnotbeensubstantiated.Toovercomethese hurdles, we have developed the PIMAX technology that produces hyperphosphorylated tau (p-tau) in E. coli. Purified p-tau fibrillizes autonomously (without an inducer), and causes apoptosis of different cells including a neuroblastomacellline.Thisinducer-free,p-tauaggregationassayhasaZ?valueof0.699,andacoefficientof variation(CV)of8.3%.Theseparametersqualifyourp-tauaggregationassayasarobustHTSplatform.Using thisassay,weconductedapilotscreenfor1,280compoundsfortheirabilitytomodulatetheaggregationofp- tau.Wethenusednovelbiochemicalandcell-basedsecondaryassaystoverifythecandidatecompounds.We foundthatanactiveneurologicaldrugisapotentp-tauaggregationinhibitor,whichalsoprotectscellsfromp- tau cytotoxicity. These preliminary studies afford solid evidence for the values of p-tau in the quest for AD therapeutics.ThisR01projectistheveryfirstthatusesthepathophysiologicallyrelevanthyperphosphorylated tau for AD drug discovery. We will follow our pilot screen protocols to conduct a high-throughput screen of 100,000compoundsfortheirabilitytoinhibitp-tauaggregationandtoprotectcells.Thisearlydiscoveryproject willconcludewithtwomajorproducts:(1)Acollectionofchemicalhitswithconfirmedp-tauaggregationinhibitory and cytoprotective activities, and (2) a follow-up proposal with comprehensive plans to identify the optimal chemicalleadforADdrugdevelopment.